Though DNase does not contain any cysteine residues, incubation of the enzyme with 2-nitro-5-thiocyanobenzoic acid (NTCB) in the presence of Ca2 ion at pH above 7.5 results in an irreversible inactivation of the enzyme. The inactivation also occurs when Ca2 ion is replaced by Mg2 ion, but not in their absence. The inactivation rate is pseudo-first order and the time to reduce half of the enzymatic activity is approximately 30 min in 25 mM NTCB and 10 mM CaC12 at pH 8.5. Gel permeation chromatography (Sephadex G-100) of NTCB-treated DNase shows aggregation of the protein in NH4HCO3 buffer. However, sodium dodecyl sulfate gel electrophoresis shows cleavages of the enzyme; fragments of molecular weights of 25,000, 28,000, 13,000 and 11,000 were observed. The reagent, under conditions where bovine DNase is inactivated, also inactivates malt DNase; it, however, does not inactivate other nucleases such as bovine ribonuclease and snake venom phosphodiesterase. Ovine pancreatic deoxyribonuclease (DNase) has been purified to apparent homogeneity. The extraction and (NH4)2SO2 fractionation procedure was essentially the same as that of Kunitz for bovine DNase. The 10-40% fraction of (NH4)2SO4 was then chromatographed at pH 4.7 on CM-cellulose which separates DNase into two active fractions. Both fractions again showed two types of DNase as each one was passed through Con-A-Agarose columns; one of them was not retained by Con-A-Agarose, while the other was bound to Con A-Agarose and could be eluted by 5% glucose. All four types of DNase appeared homogeneous on polyacrylamide gel electrophoresis.